重组半胱氨酸脱巯基酶AtLCD的表达、纯化及酶学性质

中国生物工程杂志

2013, Vol. 33

Issue (11): 8-13

研究报告

重组半胱氨酸脱巯基酶AtLCD的表达、纯化及酶学性质

袁慧虹¹, 梁雅丽^1,2, 沈洁洁¹, 张丽萍¹, 刘志强¹, 裴雁曦¹

1 山西大学生命科学学院太原 030006;
2 山西省生物研究所太原 030006

Expression, Purification and Enzymatic Characterization of Arabidopsis L-Cysteine Desulfhydrase

YUAN Hui-hong¹, LIANG Ya-li^1,2, SHEN Jie-jie¹, ZHANG Li-ping¹, LIU Zhi-qiang¹, PEI Yan-xi¹

1 School of Life Science Shanxi University, Taiyuan 030006, China;
2 Biology Institute of Shanxi, Taiyuan 030006, China

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摘要： 目的：在重组大肠杆菌中表达重组拟南芥L-半胱氨酸脱巯基酶AtLCD（H₂S内源产生关键酶），确定最佳诱导表达和纯化条件，并研究重组酶的酶学性质。方法：对大肠杆菌BL21 （DE3）/pET28a-LCD进行异丙基-β-D-硫代半乳糖苷（IPTG）诱导培养，将纯化的目的蛋白AtLCD进行酶学性质的研究。结果：重组蛋白的最佳诱导表达条件为0.1 mmol/L IPTG，30℃诱导3 h。500 mmol/L的咪唑洗脱Ni柱可得较纯的目的蛋白。重组酶性质研究结果表明，酶的最适pH为9.5，最适作用温度为37℃，最适条件下的以L-Cys为底物的米氏常数（Km）为1.572 mmol/L，Vmax为1.52 nmol/mg·min。金属离子Mg²⁺，Fe³⁺和EDTA对重组酶的活性有一定的抑制作用，Ba²⁺、Ca²⁺和Co²⁺在一定程度上提高了酶的活性，其中Co²⁺效果更强。蛋白变性剂SDS和酶抑制剂羟胺也不同程度地抑制了酶的活性。结论：明确了重组AtLCD的酶学性质和最佳诱导表达及纯化条件，为该LCD的深入研究和气体信号分子H₂S在植物应答逆境胁迫机制的研究奠定了基础。

关键词： 硫化氢; AtLCD; 酶学性质

Abstract: Objective: The recombinant Arabidopsis thaliana L-Cysteine Desulfhydrase (AtLCD) that catalyze the generation of endogenous H₂S was expressed in recombinant E. coli BL21 (DE3)/pET28a-LCD strains. We characterized a AtLCD from E. coli and optimized its the condition of induction and purification. Methods: AtLCD were purified from E. coli BL21 (DE3)/pET28a-LCD to analyze its enzymatic properties after it was induced by isopropyl-b-D-thiogalactopyranoside (IPTG). Results: The optimized conditions were: the AtLCD protein was induced by 0.1 mmo/L IPTG at 30℃ for 3 h, and purified through Ni–AKTA column, and the purified AtLCD of optimized imidazole was 500 mmol/L. The results shows that the Optimal pH value and temperature of the AtLCD were 9.5 and 37℃, respectively. Under the optimum conditions, Km value and Vmax of the AtLCD for hydrolysis of L-Cysteine was 1.572 mmol/L and 1.52 nmol/ (mg·min). Effects of metal ions on the activity of recombination AtLCD showed that Mg²⁺, Fe³⁺ and EDTA inhibited the enzyme activity lightly, while Ba²⁺, Ca²⁺and Co²⁺enhanced the enzyme activity, Co²⁺enhanced it obviously. Moreover, the recombination AtLCD activity was inhibited varying degrees by SDS and hydroxylamine. Conclusion: Enzymatic properties of AtLCD was obtained and optimized its the condition of induction and purification. It will provide theoretical basis for AtLCD and response to adversity stress mechanism of an important gasotransmitter H₂S in plants.

Key words: Hydrogen sulfide L-Cysteine Desulfhydrase Enzymatic properties

收稿日期: 2013-08-07 出版日期: 2013-11-25

ZTFLH:

Q342

基金资助: 国家自然科学基金（31372085）、山西省回国留学人员（2011-007）资助项目

通讯作者: 裴雁曦 E-mail: peiyanxi@sxu.edu.cn

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引用本文:

袁慧虹, 梁雅丽, 沈洁洁, 张丽萍, 刘志强, 裴雁曦. 重组半胱氨酸脱巯基酶AtLCD的表达、纯化及酶学性质[J]. 中国生物工程杂志, 2013, 33(11): 8-13.

YUAN Hui-hong, LIANG Ya-li, SHEN Jie-jie, ZHANG Li-ping, LIU Zhi-qiang, PEI Yan-xi. Expression, Purification and Enzymatic Characterization of Arabidopsis L-Cysteine Desulfhydrase. China Biotechnology, 2013, 33(11): 8-13.

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I11/8

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